ATCC® CRL-2876 ™ may take 24 to 48 hours to attach during recovery and after subcultures. The cells grow very slowly.
Under conditions used at ATCC, a T75 flask set up was 20-30% confluent after 3 days in culture. The cells were left in the same vessel for a total of 2 weeks, at which time they reached only 40-50% confluency, with a dense mixture of adherent cells, floating clusters and some debris. A 1:3 split ratio took another two weeks to reach the same level of confluence with a similar appearance.
The cells may recover a little better in a T-25 set-up. Under conditions used at ATCC, when one ampoule was seeded to a T-25 flask with 10 mL complete growth medium, it took about a week to reach 60-70% confluence.
The cells are small and form very tight clusters that can be broken apart with repeated pipetting. As the cells attach and spread out, there will be a mixture of flattened epithelial-like islands interspersed with the cell clusters. Do not discard the floating clusters of cells. Instead, spin them down with fluid changes and add them back to the parent flask by pipetting well in fresh medium. Observance of moderate to heavy debris is very normal.
It is highly recommended that good, high quality serum (ATCC Catalog number 30-2020) and a DMEM formulation that is very close, if not identical, to the ATCC formulation (Catalog number 30-2002) be used for these cells. The use of DMEM formulations with a high level of sodium bicarbonate (3.7 g/L) are not suitable for this cell line.
|Date Created||01/17/2014 10:28 AM
|Date Updated||01/30/2014 08:00 AM